K8000 envision flex

All immunostaining was carried out at room temperature using DAKO Autostainer Universal Staining System (Autostainer Link 48 DAKO, Glostrup, Denmark). First, sections 4 μm in thickness obtained from selected paraffin embedded blocks were put on positively charged slides. Second, all the sections were deparaffinized in xylene and dehydrated through a graded series of ethanol solution. Third, antigen retrieval was performed at 96°C (10 mM/L citrate buffer, pH 6) for 40 minutes in a thermostatic bath (PT Link). The sections were incubated with NF-κB/p50 (RB-1648; NeoMarkers, Fremont, CA, USA), MMP-1 (MS-1687-R7 NeoMarkers, CA, USA), anti-Ki-67 monoclonal antibody MIB-1 (M724029, DAKO, Glostrup, Denmark), and anti-p53 (M700129, DAKO, Glostrup, Denmark) for 60 minutes at room temperature. Positive and negative controls were added for each antibody and to each batch of staining. A streptavidin-biotin enhanced immunoperoxidase technique (K8000 Envision Flex, DAKO, Glostrup, Denmark) in an automated system was used to show immunoreactions. The sections were incubated with DAB and counterstained lightly with haematoxylin to demonstrate binding. Finally, the sections were dehydrated and mounted onto the slides. The slights known to be positively immunostained were used as positive controls. Normal rabbit serum IgG was used to replace primary antibody as a negative control.

All immunostaining was carried out at room temperature by using a DAKO Autostainer Universal Staining System (Autostainer Link 48 DAKO, Glostrup, Denmark). First, sections 4 μm in thickness obtained from selected paraffin-embedded blocks were put on positively charged slides. Second, all the sections were deparaffinized in xylene and dehydrated through a graded series of ethanol solution. Third, antigen retrieval was performed at 96°C (10 mM/L citrate buffer, pH 6) for 40 minutes in a thermostatic bath (PT Link). The sections were incubated with PDL-1 (PDL-1 IHC 22C3 pharmDx, code SK006, DAKO, Glostrup, Denmark) and MHC-1 (HLA-ABC clone W 6/32, code R7000, DAKO, Glostrup, Denmark) for 60 minutes at room temperature. Positive and negative controls were added for each antibody and to each batch of staining. A streptavidin-biotin-enhanced immunoperoxidase technique (K8000 Envision Flex, DAKO, Glostrup, Denmark) in an automated system was used to show immunoreactions. The sections were incubated with DAB and counterstained lightly with hematoxylin to demonstrate binding. Finally, the sections were dehydrated and mounted onto the slides. The tissues known to be positively immunostained were used as positive controls. Normal rabbit serum IgG was used to replace a primary antibody as a negative control.